Efficient, rapid, save and cost efficient purification of mammalian IgG antibodies, in particular human and/or humanized IgG antibodies is a much studied problem in the art. With the advent of new antibody based medicaments, purification of IgG becomes a more and more critical and costly step in the production of antibody based medicaments, requiring a high degree of purity. In addition, such antibodies must retain binding affinity and biological activities like effector functions.
For the purification of mammalian IgG antibodies, in particular human IgG or humanized IgG antibodies, commonly used purification methods comprise the use of classical biochemical separation and purification techniques such as anion/kation exchange, size-exclusion/gelfiltration, precipitations and use of specific affinity ligands. Commonly used ligands are bacterially derived proteins, Protein-A and Protein-G. Alternatively, Protein L can be used, but only for those immunoglobulins comprising a kappa light chain since Protein L does not bind lambda light chains.
Protein-A is a bacterial surface protein expressed by Staphylococcus aureus. Protein-A primarily recognizes a common site at the interface between CH2 and CH3 domains on the Fc part of human IgG1, IgG2 and IgG4 antibodies (Fcγ). In addition, Protein-A also shows binding to 12% of mouse and 50% of human VH domains (human VH-III subclass). Although these latter interactions have a lower affinity (±200 nM for VH compared to <1 nM for e.g. human IgG1) Protein-A can be used for purification of Fab—and (sc)Fv fragments (independent of the Ig isotype). Protein-A, like Protein L acts as a superantigen on human B lymphocytes, probably induced by its VH-III reactivity. Therefore, if the purified IgG antibodies are intended for therapeutic usage, a major safety concern is the possible presence of Protein-A in the purified therapeutic product as a result from the unintended detachment of Protein-A from its support material during the purification process (Protein-A leakage). Numerous publications link Protein-A with toxicity and mitogenicity in animal models and humans (see, for example, Bensinger et al., J. Biol. Resp. Modif. 3,347, 1984; Messerschmidt et al., J. Biol. Resp. Modif. 3,325, 1984; Terman and Bertram, Eur. J. Cancer Clin. Oncol. 21, 1115; 1985; and Ventura et al., Hortobagyl. Cancer Treat Rep. 71,411, 1987).
Furthermore, co-binding of Protein-A to human VH-III domains is the main reason for causing elution pH differences in affinity chromatography amongst several IgG antibodies. Such differences are not desirable because it causes a lack of consistency in purification procedures among different monoclonal antibodies (Mabs). Furthermore, tightly bound IgG Mabs, due to co-binding of Protein-A to human VH-III, often require a lower pH value of the eluents in order to obtain efficient recoveries.
Protein-G is a bacterial surface protein expressed by group C and G streptococci. Like Protein-A, Protein-G also recognizes a common site at the interface between CH2 and CH3 domains on the Fc part of human IgG1, IgG2, IgG3 and IgG4 antibodies (Fcγ). Compared to Protein-A, a broader range of IgG species can be recognized. In addition, Protein-G shows binding to the Fab portion of IgG antibodies through binding to the CH1 domain of IgG. Binding affinity towards CH1 (±200 nM) is again significantly lower compared to its epitope on the Fc part. Although Protein-G has a wider reactivity profile than Protein-A, the binding of antibodies to Protein-G is often stronger, making elution and complete antibody recovery more difficult.
The most commonly used ligand for affinity purification of human immunoglobulins, in particular IgG's, for large-scale process applications is Protein-A. However, protein-A lacks the capability of binding to human antibodies of the IgG3 subclass. In addition, Protein-A and G strongly bind to the CH2-CH3 interface on the Fc portion of IgG antibodies. Experimental data indicate that induced fit occurs, which may explain the harsh conditions required for elution. These harsh conditions may affect the conformation of the binding sites, thereby altering the immune function of purified IgG antibodies (P. Gagnon, 1996, in Purification tools for monoclonal antibodies, published by Validated Biosystems, Inc 5800N). X-ray crystallographic measurements have shown that through binding to Protein-A, the CH2 domains can be displaced longitudinally towards the CH3 domains, which finally causes partial rotation and destabilization of the carbohydrate region between the CH2 domains. The distortion interferes with subsequent protein-protein interactions that are required for the IgG to exert its effector functions. Aside from the consequences of harsh elution conditions (especially for Protein-G) on the antigen binding capabilities, these secondary effects sometimes interfere with or alter antibody effector functions and increased susceptibility of immunoglobulins to proteolysis. Loss of effector functions, caused by denaturation, altered folding and chemical modifications that arise during purification steps, are highly undesirable if the human or humanized antibodies are to be used for therapeutic purposes. In particular, reduction of intra- and inter-molecular sulphur bridges is often a problem that arises during purification and storage.
As alternative to human IgG binding proteins like Protein-A and G, several mouse monoclonal antibodies (Mabs) have been described in literature that are capable of binding to the Fc domain of human IgG antibodies. (Nelson P N, et al. Characterisation of anti-IgG monoclonal antibody A57H by epitope mapping. Biochem Soc Trans 1997; 25:373.)
Some common Fc epitopes have been identified and a number of examples are listed below: Mabs G7C, JD312 have a binding epitope on CH2, amino acids 290-KPREE-294. Mabs PNF69C, PNF110A, PNF211C, have a binding epitope on CH2-CH3, AA: 338-KAKGQPR-344. Mab A57H shows binding epitope on CH3, AA 380-EWESNGQPE-388. A problem associated with the use of mouse monoclonals, or monoclonals from other non-human species, is the release of Mabs from the matrix which causes contamination in the purified preparations that is difficult to remove. Furthermore, monoclonal antibodies and functional fragments thereof (Fab, Fab2) are easily denatured and S—S bridges, keeping the 3D structure of the molecule and the heavy and light chain aligned, are easily disrupted, in particular under harsh elution conditions that are oftentimes required for release of column bound human IgG's. Due to the vulnerability of the affinity ligands the capacity of the column is rapidly reduced, and columns have a very limited re-use capacity after elution and are unsuitable for continuous operation.
Instead of (sc)Fv fragments as described in EP-A-434317, antibody fragments derived from antibodies naturally devoid of light chains (VHH) as described in WO2006/059904 can also be used to generate immunosorbent materials for the purification of human IgG antibodies. Advantage of use of these VHH fragments are that they are single domain peptides, which are exceptionally stable even at higher temperatures. Furthermore, VHH's, are small and easily produced in cost-efficient host organisms such as Saccharomyces cerevisiae. In addition, due to the sequence similarity between these VHH fragments and the human VH-III domain family, immunogenecity is expected to be very low compared to bacterial surface proteins like Protein-A and G. These antibodies are described in more detail in EP-A-656946.
However, the amino acid sequences as described in WO2006/059904 relate to VHH fragments that bind to the light chain of human antibodies of either the kappa or lambda isotype, and as such do not enable selective purification of antibodies of the IgG isotype only.